Pharmacogenetics: the ethical issues.
Explore the source record for details and available documents.
Confidentiality↗
Biomedical subjects
P Lipton
Publications and source records attributed to P Lipton.
Do active cerebral neurons really use lactate rather than glucose?
Glucose has long been considered the substrate for neuronal energy metabolism in the brain. Recently, an alternative explanation of energy metabolism in the active brain, the astrocyte-neuron lactate shuttle hypothesis, has received attention. It suggests that during neural activity energy needs in glia are met by anaerobic glycolysis, whereas neuronal metabolism is fueled by lactate released from glia. In this article, we critically examine the evidence supporting this hypothesis and explain, from the perspective of enzyme kinetics and substrate availability, why neurons probably use ambient glucose, and not glial-derived lactate, as the major substrate during activity.
Animals↗
Can the hippocampus tell time? The temporo-septal engram shift model.
An essential feature of episodic memory, the type of memory dependent on hippocampus, is that individual memories belong to particular moments in time. Recent PET studies suggest that memory encoding and recall occur at different locations in human hippocampus. Coupled with other attributes of hippocampus, this suggested to us that the septo-temporal hippocampal axis may play an important role in time perception. We propose a temporo-septal engram shift model of hippocampal memory. The model posits that memories gradually move along the hippocampus from a temporal encoding site to ever more septal sites from which they are recalled. We propose that the sense of time is encoded by the location of the engram along the temporo-septal axis.
Hippocampus↗
Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondria.
This work determined Ca2+ transport processes that contribute to the rise in cytosolic Ca2+ during in vitro ischemia (deprivation of oxygen and glucose) in the hippocampus. The CA1 striatum radiatum of rat hippocampal slices was monitored by confocal microscopy of calcium green-1. There was a 50-60% increase in fluorescence during 10 min of ischemia after a 3 min lag period. During the first 5 min of ischemia the major contribution was from Ca2+ entering via NMDA receptors; most of the fluorescence increase was blocked by MK-801. Approximately one-half of the sustained increase in fluorescence during 10 min of ischemia was caused by activation of Ca2+ release from mitochondria via the mitochondrial 2Na+-Ca2+ exchanger. Inhibition of Na+ influx across the plasmalemma using lidocaine, low extracellular Na+, or the AMPA/kainate receptor blocker CNQX reduced the fluorescence increase by 50%. The 2Na+-Ca2+ exchange blocker CGP37157 also blocked the increase, and this effect was not additive with the effects of blocking Na+ influx. When added together, CNQX and lidocaine inhibited the fluorescence increase more than CGP37157 did. Thus, during ischemia, Ca2+ entry via NMDA receptors accounts for the earliest rise in cytosolic Ca2+. Approximately 50% of the sustained rise is attributable to Na+ entry and subsequent Ca2+ release from the mitochondria via the 2Na+-Ca2+ exchanger. Sodium entry is also hypothesized to compromise clearance of cytosolic Ca2+ by routes other than mitochondrial uptake, probably by enhancing ATP depletion, accounting for the large inhibition of the Ca2+ increase by the combination of CNQX and lidocaine.
Animals↗
Autoradiographic measurements of protein synthesis in hippocampal slices from rats and guinea pigs.
Protein synthesis is an extremely important cell function and there is now good evidence that changes in synthesis play important roles both in neuronal cell damage from ischemic insults and in neural plasticity though the mechanisms of these effects are not at all clear. The brain slice, and particularly the hippocampal slice, is an excellent preparation for studying these effects although, as with all studies on slices, caution must be exercised in that regulation in the slice may be different from regulation in vivo. Studies on neural tissue need to take into account the heterogeneity of neural tissue as well as the very different compartments within neurons. Autoradiography at both the light and electron microscope levels is a very powerful method for doing this. Successful autoradiography depends on many factors. These include correct choice of precursor amino acid, mechanisms for estimating changes in the specific activity of the precursor amino acid pool, and reliable methods for quantitation of the autoradiographs. At a more technical level these factors include attention to detail in processing tissue sections so as to avoid light contamination during exposure and developing and, also, appropriate choices of the various parameters such as exposure time and section thickness. The power of autoradiography is illustrated here by its ability to discern effects of ischemia and of plasticity-related neural input on distinct cell types and also in distinct compartments of neurons. Ischemia inhibits protein synthesis in principal neurons but activates synthesis in other cell types of the brain slice. Plasticity-related neural input immediately enhances protein synthesis in dendrites but does not affect cell bodies.
Animals↗
Ischemic cell death in brain neurons.
This review is directed at understanding how neuronal death occurs in two distinct insults, global ischemia and focal ischemia. These are the two principal rodent models for human disease. Cell death occurs by a necrotic pathway characterized by either ischemic/homogenizing cell change or edematous cell change. Death also occurs via an apoptotic-like pathway that is characterized, minimally, by DNA laddering and a dependence on caspase activity and, optimally, by those properties, additional characteristic protein and phospholipid changes, and morphological attributes of apoptosis. Death may also occur by autophagocytosis. The cell death process has four major stages. The first, the induction stage, includes several changes initiated by ischemia and reperfusion that are very likely to play major roles in cell death. These include inhibition (and subsequent reactivation) of electron transport, decreased ATP, decreased pH, increased cell Ca(2+), release of glutamate, increased arachidonic acid, and also gene activation leading to cytokine synthesis, synthesis of enzymes involved in free radical production, and accumulation of leukocytes. These changes lead to the activation of five damaging events, termed perpetrators. These are the damaging actions of free radicals and their product peroxynitrite, the actions of the Ca(2+)-dependent protease calpain, the activity of phospholipases, the activity of poly-ADPribose polymerase (PARP), and the activation of the apoptotic pathway. The second stage of cell death involves the long-term changes in macromolecules or key metabolites that are caused by the perpetrators. The third stage of cell death involves long-term damaging effects of these macromolecular and metabolite changes, and of some of the induction processes, on critical cell functions and structures that lead to the defined end stages of cell damage. These targeted functions and structures include the plasmalemma, the mitochondria, the cytoskeleton, protein synthesis, and kinase activities. The fourth stage is the progression to the morphological and biochemical end stages of cell death. Of these four stages, the last two are the least well understood. Quite little is known of how the perpetrators affect the structures and functions and whether and how each of these changes contribute to cell death. According to this description, the key step in ischemic cell death is adequate activation of the perpetrators, and thus a major unifying thread of the review is a consideration of how the changes occurring during and after ischemia, including gene activation and synthesis of new proteins, conspire to produce damaging levels of free radicals and peroxynitrite, to activate calpain and other Ca(2+)-driven processes that are damaging, and to initiate the apoptotic process. Although it is not fully established for all cases, the major driving force for the necrotic cell death process, and very possibly the other processes, appears to be the generation of free radicals and peroxynitrite. Effects of a large number of damaging changes can be explained on the basis of their ability to generate free radicals in early or late stages of damage. Several important issues are defined for future study. These include determining the triggers for apoptosis and autophagocytosis and establishing greater confidence in most of the cellular changes that are hypothesized to be involved in cell death. A very important outstanding issue is identifying the critical functional and structural changes caused by the perpetrators of cell death. These changes are responsible for cell death, and their identity and mechanisms of action are almost completely unknown.
Animals↗
Mechanism of glutamate release from rat hippocampal slices during in vitro ischemia.
There was a large release of endogenous glutamate and of pre-accumulated [3H]-D-aspartate from rat hippocampal slices during deprivation of oxygen and glucose (in vitro ischemia). The role of Na(+)-dependent glutamate transporters in this process was investigated. The release of both glutamate and [3H]-D-aspartate was largely blocked by two competitive substrate analogues of the Na(+)-dependent glutamate transporters (L-trans-pyrrolidine-2,4-dicarboxylate and D,L-threo-B-hydroxyaspartate) if the substrate analogues were intracellularly loaded prior to the ischemia. The pre-loaded analogue, D,L-threo-B-hydroxyaspartate, did not block exocytotic release of glutamate, induced by high-potassium. Dihydrokainate, an inhibitor of a subset of the Na(+)-dependent transporters, did not inhibit ischemia-induced release of glutamate or [3H]-D-aspartate. However, it did block release induced by veratridine, which was also blocked by the pre-loaded substrate analogues. Dihydrokainate could still inhibit veratridine-induced release during ischemia, showing that conditions during ischemia did not reduce its efficacy. It is concluded that release of glutamate during ischemia is largely via reversal of the Na(+)-dependent glutamate transport system. The differential effects of dihydrokainate and the competitive substrate analogues on ischemia-induced release indicate that this release occurs via a subset of the glutamate transporters that are present in the hippocampus.
Animals↗
Preparative methods for brain slices: a discussion.
Criteria for slice health and factors that affect slice health were discussed by many of the participants in the conference. In addition to the standard parameters of slice health (energy metabolism, morphology, electrophysiological responsiveness) more subtle but possibly equally important manifestations of slice health were discussed. These included protein synthesis, and more subtle changes, of which we are becoming increasingly aware. The latter include synthesis of stress-related proteins, altered levels of phosphorylation, altered levels of proteolysis. These last were only touched on, but it is becoming apparent they do in fact constitute important manifestations of differences between the slice preparation and the in vivo tissue. They may well lead to quite different responses in slices from those that occur in vivo. While many ways of optimizing slice wellness were discussed, there was a fair consensus that certain adjustments will optimize the most widely measured aspects of cell function. These include the following, wherever possible. Use of young animals, use of the interface chamber, preparing slices with the vibratome, pre-treating animals with ice-cold cardiac perfusion before sacrificing, using pre-incubation media which reduce NMDA receptor activation, free radical formation and cell swelling. When possible these treatments should perhaps be continued into the normal incubation. This being said, many viewpoints were actually expressed in the discussion, and it should be read to get a feel for the usefulness of the different approaches.
Animals↗
Making the best of brain slices: comparing preparative methods.
Explore the source record for details and available documents.
Animals↗
Pairing the cholinergic agonist carbachol with patterned Schaffer collateral stimulation initiates protein synthesis in hippocampal CA1 pyramidal cell dendrites via a muscarinic, NMDA-dependent mechanism.
Effects of afferent stimulation on local synthesis of protein in CA1 pyramidal cell dendrites were studied using light microscope autoradiography. Tissue was fixed with paraformaldehyde immediately after 3 min exposure to 3H-leucine in order to trap 3H associated with macromolecules. The rate of 3H-leucine incorporation into dendrites of resting hippocampal slices was 10% the rate of incorporation into cell somata. Ninety percent of the incorporation into the somata was inhibited by cycloheximide (300 microM); none of the incorporation into dendrites was blocked by cycloheximide. Thus, there is no measurable extramitochondrial synthesis of protein in the dendrites of the resting slice. Slices were exposed to 50 microM carbachol and the Schaffer collateral afferents to the CA1 pyramidal cells were stimulated intermittently at 10 Hz over a 20 min period. In this case, 3H incorporation into dendrites was increased almost threefold over resting levels, with no effect on label over the cell somata. There was no associated increase in uptake of free 3H-leucine, and the increase in label was completely blocked by cycloheximide. Thus, associating carbachol and afferent stimulation appears to activate de novo protein synthesis in the dendrites. Neither the carbachol alone nor the Schaffer collateral stimulation alone increased synthesis. The activation of dendrite synthesis was completely blocked by 5 microM atropine, and also by 50 microM D-aminophosphonovalerate. It did not occur when carbachol was paired with steady stimulation of the Schaffer collaterals at 1 Hz for 20 min, rather than with the patterned high-frequency stimulation. Thus, associating a cholinergic agonist with a level of neural activity that occurs in CA3 and CA1 pyramidal cells during exploratory behavior (Muller et al., 1987) initiates local protein synthesis in target dendrites. This effect is dependent on muscarinic cholinergic receptors and NMDA-type glutamate receptors. The possible relationship of this phenomenon to mechanisms of learning is discussed.
2-Amino-5-phosphonovalerate↗
Intracellular calcium levels and calcium fluxes in the CA1 region of the rat hippocampal slice during in vitro ischemia: relationship to electrophysiological cell damage.
Five minutes of oxygen and glucose deprivation (termed "in vitro ischemia") causes long-term synaptic transmission failure (LTF) in the CA1 region of the rat hippocampal slice. Dependence of LTF on cell calcium was tested by generating graded reductions in cell Ca. There was a strong correlation between the average level of exchangeable cell Ca in CA1 during ischemia, and the extent of LTF. In standard buffer, exchangeable cell Ca in CA1 increased by 35% after 3 min of ischemia and remained elevated for the entire 5 min of ischemia. Unidirectional Ca influx increased by 35% during the first 2.5 min of ischemia and remained at that level for the next 2.5 min. There were no changes in unidirectional Ca efflux during this period. Thus, the accumulation results from increased influx of Ca. Ca influx during the first 2.5 min of ischemia depended entirely on NMDA channels; it was completely blocked by the noncompetitive NMDA receptor antagonist MK-801. However MK-801 had no effect during the second 2.5 min. This inactivation of NMDA-mediated influx during ischemia appears to result from dephosphorylation. Okadaic acid increased Ca influx during the second 2.5 min of ischemia and this increase was blocked by MK-801. The ischemia-induced Ca influx during the second 2.5 min of ischemia was attenuated 25% by nifedipine (50 microM) and an additional 35% by the Na/Ca exchange inhibitor benzamil (100 microM). The AMPA/kainate antagonist DNQX had no effect on the Ca influx. Antagonists were used to relate Ca influx to LTF. Blockade of enhanced Ca entry during ischemia in standard buffer (2.4 mM Ca) had no effect on LTF, consistent with total cell Ca prior to ischemia being adequate to cause complete LTF. However, MK-801 strongly protected against LTF when the buffer contained 1.2 mM Ca, a more physiological level. MK-801 combined with DNQX prevented transmission damage in standard buffer. Thus, AMPA/kainate receptor activation contributes to ischemic damage, although not by enhancing Ca entry.
Amiloride↗
Sigma-ligands and non-competitive NMDA antagonists inhibit glutamate release during cerebral ischemia.
Release of glutamate from brain cells is increased during ischemia and is thought to be involved in ischemic damage. In rat hippocampal slices the release of glutamate during 'in vitro ischemia' (anoxia without glucose) is shown to be blocked by two groups of compounds: non-competitive N-methyl-D-aspartate (NMDA) antagonists and sigma ligands. The effects are selective for the ischemic glutamate release, which is independent of extracellular Ca2+. High K+, Ca2+ dependent, induced release of glutamate is not inhibited. NMDA receptor blockade normally does not prevent ischemic transmission damage in the rat hippocampal slice. However, when ischemic glutamate release is attenuated, NMDA receptor antagonists do prevent the damage. This indicates that high levels of glutamate may cause damage via non-NMDA as well as NMDA receptors.
2-Amino-5-phosphonovalerate↗
In vitro ischemia and protein synthesis in the rat hippocampal slice: the role of calcium and NMDA receptor activation.
The rat hippocampal slice was developed as a model for investigating the effects of ischemia on protein synthesis in different cell types, as synthesis is an early functional indicator of cell damage. Five min of in vitro ischemia inhibited protein synthesis in CA1 pyramidal and subicular neurons 3 h later, despite recovery of the energy charge. Morphology of these neurons was also affected. In contrast, glia and capillary endothelial cells showed increased synthesis at this time point, and no apparent structural changes. Exposure of slices to buffer lacking calcium and containing the non-competitive NMDA receptor blocker ketamine, during the 5 min ischemia, prevented both the inhibition of protein synthesis and the morphologic changes in the neurons. However, if buffer only lacked calcium, or only contained ketamine, both forms of ischemic damage occurred. Thus, the neuronal protein synthesis inhibition and the impaired morphology appear to be mediated by either extracellular calcium or NMDA receptor activation. In contrast to the neurons, the ischemia-induced stimulation of protein synthesis in glia and capillary endothelial cells was not affected by the above treatments, indicating that neither NMDA receptor activation nor extracellular calcium is necessary for this effect.
Animals↗
NMDA receptor activation accelerates ischemic energy depletion in the hippocampal slice and the demonstration of a threshold for ischemic damage to protein synthesis.
Energy depletion is a primary factor initiating ischemic damage to neurons. In a separate report, we demonstrated that in vitro ischemia inhibits protein synthesis in the CA1 pyramidal neurons of the hippocampal slice via a mechanism involving extracellular calcium and N-methyl-D-aspartate (NMDA) receptor activation during the ischemic episode. In this study, we tested whether these agents accelerated the ischemic energy depletion beyond tolerable levels. ATP and phosphocreatine (PCr) were measured immediately after different durations of in vitro ischemia in the presence or absence of calcium and the NMDA receptor antagonist ketamine. The results support the contention that extracellular calcium does not contribute to the ischemic energy depletion. NMDA receptor activation accelerates the fall in ATP and PCr, but only during the first 45 s of the ischemia. Using protein synthesis inhibition as a functional indicator of ischemic damage in the hippocampal slice, we demonstrated that greater than 2 min of ischemia is necessary to inhibit protein synthesis. Thus, this threshold duration of ischemia indicates that events occurring between 2 and 5 min ischemia result in a prolonged protein synthesis inhibition.
Adenosine Triphosphate↗
N-methyl-D-aspartate receptor activation and Ca2+ account for poor pyramidal cell structure in hippocampal slices.
The CA1 pyramidal cells appear damaged in micrographs of guinea pig hippocampal slices incubated in normal physiological buffer at 36-37 degrees C. This is remedied if slices are incubated in modified buffers for the first 45 min. Cell morphology is improved if this buffer is devoid of added Ca2+ and much improved if it contains N-methyl-D-aspartate (NMDA) receptor antagonists or 0 mM Ca2+ and 10 mM Mg2+. The cells then appear similar to CA1 pyramidal cells in situ. These findings support the notion that NMDA receptor activation and Ca2+, acting in the period immediately after slice preparation, permanently damage CA1 pyramidal cells in vitro.
Adenosine Triphosphate↗
Mechanisms of intracellular calcium accumulation in the CA1 region of rat hippocampus during anoxia in vitro.
There is a net movement of calcium into brain cells during anoxia and ischemia. This communication examines the mechanisms of this movement in rat hippocampal slices by analyzing changes in 45Ca2+ distribution. The CA1 pyramidal cells are the most sensitive to anoxic/ischemic damage; therefore, our measurements of Ca2+ and high-energy nucleotides are restricted to this region. The increase in intracellular Ca2+ levels during anoxia is not blocked by the Ca2+ channel blocker cobalt, nor is it blocked by N-methyl-D-aspartate receptor antagonists kynurenic acid, D-2-amino-5-phosphonovaleric acid, or ketamine. Kinetic measurements show that the rate of Ca2+ efflux across the plasmalemma during anoxia is sufficiently decreased to account for the increase in intracellular Ca2+. It thus appears that the net increase in calcium does not result from the opening of voltage-sensitive Ca2+ channels, nor from flux through the N-methyl-D-aspartate channel. Rather, it results from inhibition of the adenosine triphosphate-dependent extrusion mechanism for Ca2+. The relation of this conclusion to mechanisms of anoxic cell damage is discussed.
Animals↗
Regulation of glycogen in the dentate gyrus of the in vitro guinea pig hippocampus; effect of combined deprivation of glucose and oxygen.
Mammalian brain glycogen is adequate to support oxidative metabolism for several minutes. The present studies were done primarily to develop the guinea pig hippocampal slice as a model for studying the function and regulation of that glycogen. Slice glycogen falls to 6 nmol/mg dry wt. during the first hour of incubation at 36 degrees C but during the next 3 h recovers to 20 nmol/mg dry wt., similar to in situ values. Glycogen concentration in the dentate gyrus molecular layer is double its value in the whole hippocampal slice, suggesting its distribution is related to metabolic demand. When both glucose and oxygen are removed from the medium, glycogen and ATP fall to 50% within 6 min. The glycogen fall is unaffected by prolonged calcium depletion or by 3-isobutyl 1-methylxanthine, an adenosine antagonist. It is markedly slowed by preincubating the slice with creatine, which also slows the fall in ATP. It is concluded that ATP breakdown and subsequent increased 5'-AMP is activating glycogen mobilization in this in vitro model of ischemia.
1-Methyl-3-isobutylxanthine↗
Protection of hippocampal slices from young rats against anoxic transmission damage is due to better maintenance of ATP.
1. Dentate granule cells in hippocampal slices from young rats (aged 30-40 days) are more resistant to damage from 10 min of anoxia than are granule cells from adult rats. The evoked population spike from these cells recovers to 78% of its pre-anoxic amplitude in young animals while in adult animals it shows only 4% recovery. This increased resistance is associated with higher levels of adenosine triphosphate (ATP) during the anoxic period. 2. When the duration of anoxia in slices from young animals is increased to 15 min, ATP falls to levels found in adult tissue after 10 min of anoxia. The dentate granule cells in slices from young animals show little recovery of the evoked response (19%) after such an exposure to anoxia. 3. When slices from young animals are subjected to 10 min of anoxia in low-glucose (2 mM) artificial cerebrospinal fluid, ATP levels fall to those found in adult tissue after 10 min of anoxia and the evoked response from the dentate granule cells again shows little recovery (10%). 4. The evoked response in the CA1 pyramidal cell layer of slices from young rats is more resistant to damage from 5 or 7 min anoxia than it is in slices from adults. Thus this region, also, shows an age-dependent increase in susceptibility to anoxic damage. ATP levels in the CA1 region of tissue from young animals at the end of 5 and 7 min anoxia are greater than ATP levels in tissue from adult animals after these same anoxic exposures. 5. Basal levels of 45Ca accumulation are greater in CA1 and dentate gyrus from young rats. However, the percentage increases during 10 min of anoxia are less than one-half the values in slices from adult animals. 6. The results suggest that the increased resistance of slices from young animals to anoxic transmission damage may be explained by the better maintenance of ATP in synaptic regions of these slices during anoxia. This may confer the increased resistance by lowering the anoxic increase in cell Ca2+.
Adenosine Triphosphate↗